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The primary structure of duck egg-white lysozyme II
178
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
BBA
NOTES
31068
The primary structure of duck egg-white lysozyme II
....
Since the establishment of the primary structure of hen egg-white lysozyme (EC 3.2.i.i7) 1-3, the purification and some properties of m a n y lysozymes from bird egg whites, from human origin, from plants, from bacteria and from phages have been described (for a general review, see ref. 4). However, only the primary structures of two phage lysozymes have been established in detail 5, and preliminary reports concerning human lysozymes have been published6, 7. I t is astonishing that the primary structure of egg-white lysozyme of other birds was not established until this year despite m a n y comparative chiefly immunologicalS, 9 studies. A partial explanation can be found in the fact that m a n y studies were devoted to the goose enzyme, which is very labile and quite different from hen lysozyme and to the lysozymes of the duck egg-white which contains, even in the case of a well defined species, at least two enzymes 4. LARuE AND SPECK10 have just published the structure of turkey egg-white TABLE I PRIMARY STRUCTURE LYSOZYME
OF
DUCK
EGG-WHITE
LYSOZYME
IL
COMPARISON
WITH
HEN
EGG-WHITE
F o r m u l a of JOLLIES et al. 2; residues 40-42, 58-59 and 92-93 have been revised after our comparative studies devoted to duck, guinea-hen (unpublished data) and h u m a n milkS, 15 lysozymes. For amino acids No. 46 , 48 and 57, see text. Duck II Hen
i Lys-Val-Tyr-Ser A r g - C y s - G l u L e u - A l a - A l a - A l a Met-Lys-Arg-Leu G l y Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala Ala-Ala-Met-Lys-Arg-His-Gly-
Duck II Hen
L e u - A s p - A s n - T v r - A r g - G l y - T y r - S e r Leu-Gly Asn-Trp Val-Cys A l a - A l a 17 L e u - A s p - A s n - T y r - A r g - G l y - T y r - S e r Leu-Gly A s n - T r p - V a l - C y s - A l a - A l a -
Duck II Hen
33
Duck II Hen
Gly S e r - T h r - A s p Tyr G l y - I l e - L e u G l u - I l e A s n - S e r - A r g - T r p T r p - C y s 49 G l y - S e r - T h r Asp T y r - G l y - I l e Leu G l n - I l e - A s n S v r - A r g T r p - T r p - C y s -
Duck II Hen
65 Asp-Asn-Gly-Lys-Thr P r o - G l y - S e r -Lys-Asn A la-Cys-Gly - I l e P r o - C y s Asp-Asn-Gly-Arg T h r - P r o - G l y - S e r Arg-Asn-Leu-Cys-Asn-Ile - P r o - C y s -
Duck II Hen
81 S e r - V a l - L e u - L e u - A r g - S e r - A s p I l e - T h r - G l u - A l a Val-Arg C y s - A l a - L y s Ser-Ala-Leu-Leu-Ser-Ser-Asp-Ile-Thr-Ala-Ser-Val Asn-Cys-Ala-Lys-
Duck II Hen
Arg-Ile - V a l - S e r - A s p - G l y - A s p G l y - M e t - A s n - A l a T r p - V a l - A l a - T r p - A r g 97 Lys-Ile - V a l - S e r - A s p - G l y - A s p - G l y - M e t - A s n - A l a - T r p - V a l - A l a T r p - A r g -
Xsn-Tyr-Glu Ser-Ser-Phe-Asn-Thr-Gln A l a - T h r - A s n - A r g - A s n - T h r - A s p Lys Phe-Glu-Ser A sn-Phe-Asn-Thr-Gln A l a - T h r - A s n - A r g - A s p - T h r - A s n
Duck II 113 Asn-Arg-Cys-Arg-Gly-Thr-Asp-Val-Ser -Lys-Trp Ile - A r g - G l y - C y s - A r g - L e u Hen Asn-Arg-Cys-Lys-Gly-Thr-Asp-Val-Gln-Ala-Trp I l e - A r g G l y - C y s - A r g - L e u * 7oth communication on lysozymes; 69th communication, see ref. 4. *" This note is based on a p a r t of a thesis s u b m i t t e d by one of us (J.H.) in partial fulfillment of the requirements for the degree of "Docteur as Sciences" Paris (Ao3675).
Biochim. Biophys. Acta, 2oo (197 o) 178-179
PRELIMINARY NOTES
179
lysozyme and this note is concerned with the complete primary structure of duck eggwhite lysozyme II. The structure of the N-terminal moiety of this enzyme has already been reported 4,n. Duck egg-white lysozyme II was prepared according to JOLLIES et al. 1~. 300 mg enzyme were reduced, alkylated with iodoacetic acid and submitted to a tryptic hydrolysis TM. The hydrolysate was chromatographed on a Dowex I-X2 column 13 or filtered on Sephadex G-25 (fine), and the peaks were purified using different column (Dowex 5o-X2) and paper rechromatographies. Similar experiments were achieved with a chymotryptic digest. The structures of the purified peptides were established in detail following usual methods (chiefly the Edman procedure; "dansyl"-method; carboxypeptidase digestion; chymotryptic digestion of a tryptic peptide, etc.). Table I indicates the complete primary structure of duck egg-white lysozyme II. It is similar to that of hen egg-white lysozyme; however 19 replacements have been noted for only 7 between turkey and hen lysozymes1°. Furthermore the amidation of four aspartic acid and one glutamic acid residues is different from that reported either by JOLLkS et al. 2 (residues 46, 48, 57) or by CANFIELDa (residues 57, 65, 66) for similar residues in hen lysozyme. We want also to mention the following changes: leucine (15) for histidine, the duck lysozymes being devoid of this latter amino acid ; asparagine(33) for lysine; arginine(85) for serine; arginine(93) for valine; lysine(i22) for alanine. PHILLIPS' model ~4in the space of hen lysozyme allows one to indicate that nearly all the replaced amino acids are situated at its surface. The tryptophan and cystine residues occupy similar positions in duck II and hen lysozymes, and the same situation is found for residues glutamic(35) acid and aspartic(52) acid which are essential for the catalytic activity 14. All the details concerning this study as well as the complete primary structure of duck egg-white lysozyme III (ref. 12) which is different from that of duck egg-white lysozyme II (refs. 4, 13) will be published in a forthcoming paper. This research has been achieved in the laboratory of P. Joll~s with partial financial support fo the C.N.R.S. and T.N.S.E.R.M.
Laboratory of Biochemistry, Faculty of Sciences, 96 Bd. Raspail, Paris 6 ° (France)
JACQUES HERMANN JACQUELINE JOLLES
I J. JOLLIkS AND P. JOLL~$, Compt. Rend., 253 (1961) 2773. 2 J. JOtLlkS, J. JAUREGUI-ADELL, I. BERNIER AND P. JOLL~S, Biochim. Biophys. Acta, 78 (1963) 668. 3 R. E. CANFIELD, J . Biol. Chem., 238 (1963) 2698. 4 P. JOLL~S, Angew. Chem. Intern. Ed. Engl., 8 (I969) 227. 5 A. TSUGITA AND M. INOUYE, J. Mol. Biol., 37 (1968) 2Ol. 6 J. JOEL'S AND P. JOLLIES, Bull. Soc. Chim. Biol., 5 ° (1968) 2543. 7 S. KAMMERMAN AND R. E. CANFIELD, Federation Proc., 28 (1969) 343. 8 N. ARNHEIM, E. M. PRAGER AND A. C. WILSON, J. Biol. Chem., 244 (1969) 2085. 9 N. ARNHEIM, JR. AND A. C. WILSON, J. Biol. Chem., 242 (1967) 3951. io J. N. LARuE AND J. C. SPECK, JR., Federation Proc., 28 (1969) 662. i i B. NIEMANN, J. HERMANN AND J. JOLLIkS, Bull. Soc. Chim. Biol., 5 ° (1968) 923. 12 J. JOLLIES, G. SPOTORNO AND P. JOLLIES, Nature, 2o8 (1965) 12o 4. 13 J. JOLL~S, J. HERMANN, B. NIEMANN AND P. JOLLIES, European J. Biochem., i (1967) 344. 14 D. C. PHILLIPS, Sci. Am., 215 (1966) 78 . 15 J. JOLL/~S ET P. JOLLf~S, Helv. Chim. Acta, 52 (1969) in the press.
Received October 24th, 1969 Bioehim. Biophys. Acta, 200 (197 o) 178-179
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
BBA
NOTES
31068
The primary structure of duck egg-white lysozyme II
....
Since the establishment of the primary structure of hen egg-white lysozyme (EC 3.2.i.i7) 1-3, the purification and some properties of m a n y lysozymes from bird egg whites, from human origin, from plants, from bacteria and from phages have been described (for a general review, see ref. 4). However, only the primary structures of two phage lysozymes have been established in detail 5, and preliminary reports concerning human lysozymes have been published6, 7. I t is astonishing that the primary structure of egg-white lysozyme of other birds was not established until this year despite m a n y comparative chiefly immunologicalS, 9 studies. A partial explanation can be found in the fact that m a n y studies were devoted to the goose enzyme, which is very labile and quite different from hen lysozyme and to the lysozymes of the duck egg-white which contains, even in the case of a well defined species, at least two enzymes 4. LARuE AND SPECK10 have just published the structure of turkey egg-white TABLE I PRIMARY STRUCTURE LYSOZYME
OF
DUCK
EGG-WHITE
LYSOZYME
IL
COMPARISON
WITH
HEN
EGG-WHITE
F o r m u l a of JOLLIES et al. 2; residues 40-42, 58-59 and 92-93 have been revised after our comparative studies devoted to duck, guinea-hen (unpublished data) and h u m a n milkS, 15 lysozymes. For amino acids No. 46 , 48 and 57, see text. Duck II Hen
i Lys-Val-Tyr-Ser A r g - C y s - G l u L e u - A l a - A l a - A l a Met-Lys-Arg-Leu G l y Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala Ala-Ala-Met-Lys-Arg-His-Gly-
Duck II Hen
L e u - A s p - A s n - T v r - A r g - G l y - T y r - S e r Leu-Gly Asn-Trp Val-Cys A l a - A l a 17 L e u - A s p - A s n - T y r - A r g - G l y - T y r - S e r Leu-Gly A s n - T r p - V a l - C y s - A l a - A l a -
Duck II Hen
33
Duck II Hen
Gly S e r - T h r - A s p Tyr G l y - I l e - L e u G l u - I l e A s n - S e r - A r g - T r p T r p - C y s 49 G l y - S e r - T h r Asp T y r - G l y - I l e Leu G l n - I l e - A s n S v r - A r g T r p - T r p - C y s -
Duck II Hen
65 Asp-Asn-Gly-Lys-Thr P r o - G l y - S e r -Lys-Asn A la-Cys-Gly - I l e P r o - C y s Asp-Asn-Gly-Arg T h r - P r o - G l y - S e r Arg-Asn-Leu-Cys-Asn-Ile - P r o - C y s -
Duck II Hen
81 S e r - V a l - L e u - L e u - A r g - S e r - A s p I l e - T h r - G l u - A l a Val-Arg C y s - A l a - L y s Ser-Ala-Leu-Leu-Ser-Ser-Asp-Ile-Thr-Ala-Ser-Val Asn-Cys-Ala-Lys-
Duck II Hen
Arg-Ile - V a l - S e r - A s p - G l y - A s p G l y - M e t - A s n - A l a T r p - V a l - A l a - T r p - A r g 97 Lys-Ile - V a l - S e r - A s p - G l y - A s p - G l y - M e t - A s n - A l a - T r p - V a l - A l a T r p - A r g -
Xsn-Tyr-Glu Ser-Ser-Phe-Asn-Thr-Gln A l a - T h r - A s n - A r g - A s n - T h r - A s p Lys Phe-Glu-Ser A sn-Phe-Asn-Thr-Gln A l a - T h r - A s n - A r g - A s p - T h r - A s n
Duck II 113 Asn-Arg-Cys-Arg-Gly-Thr-Asp-Val-Ser -Lys-Trp Ile - A r g - G l y - C y s - A r g - L e u Hen Asn-Arg-Cys-Lys-Gly-Thr-Asp-Val-Gln-Ala-Trp I l e - A r g G l y - C y s - A r g - L e u * 7oth communication on lysozymes; 69th communication, see ref. 4. *" This note is based on a p a r t of a thesis s u b m i t t e d by one of us (J.H.) in partial fulfillment of the requirements for the degree of "Docteur as Sciences" Paris (Ao3675).
Biochim. Biophys. Acta, 2oo (197 o) 178-179
PRELIMINARY NOTES
179
lysozyme and this note is concerned with the complete primary structure of duck eggwhite lysozyme II. The structure of the N-terminal moiety of this enzyme has already been reported 4,n. Duck egg-white lysozyme II was prepared according to JOLLIES et al. 1~. 300 mg enzyme were reduced, alkylated with iodoacetic acid and submitted to a tryptic hydrolysis TM. The hydrolysate was chromatographed on a Dowex I-X2 column 13 or filtered on Sephadex G-25 (fine), and the peaks were purified using different column (Dowex 5o-X2) and paper rechromatographies. Similar experiments were achieved with a chymotryptic digest. The structures of the purified peptides were established in detail following usual methods (chiefly the Edman procedure; "dansyl"-method; carboxypeptidase digestion; chymotryptic digestion of a tryptic peptide, etc.). Table I indicates the complete primary structure of duck egg-white lysozyme II. It is similar to that of hen egg-white lysozyme; however 19 replacements have been noted for only 7 between turkey and hen lysozymes1°. Furthermore the amidation of four aspartic acid and one glutamic acid residues is different from that reported either by JOLLkS et al. 2 (residues 46, 48, 57) or by CANFIELDa (residues 57, 65, 66) for similar residues in hen lysozyme. We want also to mention the following changes: leucine (15) for histidine, the duck lysozymes being devoid of this latter amino acid ; asparagine(33) for lysine; arginine(85) for serine; arginine(93) for valine; lysine(i22) for alanine. PHILLIPS' model ~4in the space of hen lysozyme allows one to indicate that nearly all the replaced amino acids are situated at its surface. The tryptophan and cystine residues occupy similar positions in duck II and hen lysozymes, and the same situation is found for residues glutamic(35) acid and aspartic(52) acid which are essential for the catalytic activity 14. All the details concerning this study as well as the complete primary structure of duck egg-white lysozyme III (ref. 12) which is different from that of duck egg-white lysozyme II (refs. 4, 13) will be published in a forthcoming paper. This research has been achieved in the laboratory of P. Joll~s with partial financial support fo the C.N.R.S. and T.N.S.E.R.M.
Laboratory of Biochemistry, Faculty of Sciences, 96 Bd. Raspail, Paris 6 ° (France)
JACQUES HERMANN JACQUELINE JOLLES
I J. JOLLIkS AND P. JOLL~$, Compt. Rend., 253 (1961) 2773. 2 J. JOtLlkS, J. JAUREGUI-ADELL, I. BERNIER AND P. JOLL~S, Biochim. Biophys. Acta, 78 (1963) 668. 3 R. E. CANFIELD, J . Biol. Chem., 238 (1963) 2698. 4 P. JOLL~S, Angew. Chem. Intern. Ed. Engl., 8 (I969) 227. 5 A. TSUGITA AND M. INOUYE, J. Mol. Biol., 37 (1968) 2Ol. 6 J. JOEL'S AND P. JOLLIES, Bull. Soc. Chim. Biol., 5 ° (1968) 2543. 7 S. KAMMERMAN AND R. E. CANFIELD, Federation Proc., 28 (1969) 343. 8 N. ARNHEIM, E. M. PRAGER AND A. C. WILSON, J. Biol. Chem., 244 (1969) 2085. 9 N. ARNHEIM, JR. AND A. C. WILSON, J. Biol. Chem., 242 (1967) 3951. io J. N. LARuE AND J. C. SPECK, JR., Federation Proc., 28 (1969) 662. i i B. NIEMANN, J. HERMANN AND J. JOLLIkS, Bull. Soc. Chim. Biol., 5 ° (1968) 923. 12 J. JOLLIES, G. SPOTORNO AND P. JOLLIES, Nature, 2o8 (1965) 12o 4. 13 J. JOLL~S, J. HERMANN, B. NIEMANN AND P. JOLLIES, European J. Biochem., i (1967) 344. 14 D. C. PHILLIPS, Sci. Am., 215 (1966) 78 . 15 J. JOLL/~S ET P. JOLLf~S, Helv. Chim. Acta, 52 (1969) in the press.
Received October 24th, 1969 Bioehim. Biophys. Acta, 200 (197 o) 178-179